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Researchers May Have Caught HIV Becoming More Virulent in Cuba

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A study from Cuba, recently published online in EBioMedicine, has generated wide media interest because researchers have identified a particular variety of the virus, dubbed CRF19_cpx, that is associated with rapid post-diagnosis drops in CD4 T-cell count and progression to AIDS. In the study, all of the still relatively small minority of people with this viral variant progressed to clinically defined AIDS within 3 years of infection. The variant also seems to be becoming more common in Cuba and may partly explain what appears to be an increase in the proportion of people who progress rapidly to AIDS. However, it is not a drug-resistant strain and antiretroviral therapy (ART) works just as well against it as it does against any other strain of HIV.

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This CRF19_cpx viral strain is a recombinant, which means that its genetic code is a patchwork of gene fragments that come from other varieties of HIV. In this case, the genes of CRF19_cpx come from HIV subtypes A, D, and G, which are mainly found in Africa. There, subtype D is associated with faster disease progression, and A/D recombinants even more so.

Vivian Kouri fromthe Institute of Tropical Medicine Pedro Kourí in Havana, Ricardo Khouri from KU Leuven in Belgium, and colleagues believe that the ultimate cause of CRF19_cpx's unusual virulence is that its protease gene, which comes from HIV subtype D, produces an unusually potent protease enzyme that generates exceptional numbers of viral copies and rapidly alters the arms race between HIV and the immune response against it in favor of the virus.

So-called "circulating recombinant forms" (CRFs) of HIV are not uncommon. In some countries they may even predominate, such as in Thailand, where CRF01_AE is so common it is often just called "subtype E" (this variant also has been associated with faster progression). CRF19_cpx has been found in Africa, but it is only in Cuba that it has been noted to be spreading.

It has also been noted that there appeared to be a trend towards faster progression to AIDS among people in Cuba diagnosed since 2007, with an estimated 13%-16% being true "fast progressors" (i.e., with a CD4 count below 200 cells/mm3 within 3 years of infection). This is higher than the 5%-10% reported internationally, but is not unprecedented: a study published in December found that, in Europe, the time between infection and a CD4 count below 200 cells/mm3 has halved in the last 2 decades. This may be driven by evolutionary pressure; because of antiretroviral therapy, HIV now has less time to jump between individuals and so only the fittest, fastest-replicating viruses will survive. Conversely, pressures in some parts of Africa may be driving HIV towards lower virulence in that region.

The Cuban researchers found that only 9 out of a minority of 52 people who progressed to AIDS rapidly after diagnosis had CRF19_cpx, so this virus is not the cause of all cases. But it is striking that there was no one with CRF19_cpx in this small study who was not a fast progressor. This is the first time a specific viral variant has so unambiguously been shown to be associated with rapid progression to AIDS.

The Study

Between late 2007 and 2011, patients from the Pedro Kouri Institute of Tropical Medicine, Cuba’s premier public health institute, were recruited to the study if they had been diagnosed less than 3 years after HIV infection and were not taking ART. Their CD4 count, viral load, and CD4 percentage were measured at this time. The average time between infection and diagnosis was 22 months in people who later on turned out to be slow progressors (see below) and 14 months in people who turned out to be fast progressors. This difference was not statistically significant.

The researchers waited until 36 months had elapsed from the estimated time of infection and then completed a second battery of tests that, as well as CD4 count and percentage and viral load, measured HIV subtype, the levels of a panel of different cytokines (signaling proteins of the immune system), other sexually transmitted infections, drug resistance, and also the so-called tropism of their virus. The latter is a measure of the particular immune system cells the virus likes to infect and may have profound implications for how fast people progress to an AIDS diagnosis (see more below).

On the basis of this 3-year test, 52 of the patients were included in the study as "AIDS rapid progressors," where AIDS was defined either as a CD4 count less than 200 cells/mm3, a CD4 percentage below 14%, or an AIDS-related clinical condition. A second comparison group of patients diagnosed between 2007 and 2011 who did not have any of these criteria for fast progression at the 3-year test was also recruited.

Both groups were recruited consecutively -- i.e., every diagnosed patient within a certain timeframe who fit the criteria was included -- but separately. "Fast progressors" are less common than other patients, so their group took longer to recruit, though the study was not set up to calculate what proportion of the entire clientele of the Pedro Kouri clinic were fast progressors.

If the second test showed that patients needed to start ART, as was the case with all fast progressors, then they were started on treatment at this point (Cuba’s HIV treatment guidelines still maintain 200-350 cells/mm3 as the recommended threshold for ART).

These 2 groups were compared with a third group of 22 people who met the same criteria for AIDS as the fast progressors, but who had all had HIV for more than 10 years, and where there had been at least 6 years between the HIV and AIDS diagnoses.

Test Results at Diagnosis

The 95 patients were all similar in terms of race/ethnicity, gender (80% male), age at diagnosis (average 32), and sexual behavior. The fast progressors were less likely to be gay men and considerably less likely to report anal sex.

The CD4 count at diagnosis was 577 and 522 cells/mm3, respectively, in the chronically infected and slow progressor groups, but only 276 cells/mm3 in the fast progressor group, indicating that their immune systems had suffered a particularly strong attack from HIV during acute infection.

There was a considerable difference in the first viral load measurement taken after diagnosis. In the fast progressors the median viral load was 63,000 copies/mL, in the long-term infected it was 1600 copies/mL, and in the recently diagnosed slow progressors it was undetectable, i.e., below 50 copies/mL.

This latter finding is interesting in itself: remember, none of these people were on ART. Have the researchers discovered a group of viral controllers?

Lead researcher Professor Anne-Mieke Vandamme of the Rega Institute in Leuven, Belgium told Aidsmap: "You are right to find that peculiar; we had the same concern. Half of the [slow progressor] patients had an undetectable viral load, while half had a higher viral load" (up to a few thousand copies/mL).

"We could not find any reason for that," she continued. "We double checked the data and that is just how it was in our 21 consecutively recruited patients."

In the whole group of 95 patients, 25 had HIV subtype B, 6 subtype C, 29 had B/G recombinants and other mixtures, 7 had a recombinant called CRF18_cpx, and 9 had the CRF19_cpx recombinant. All of those 9 were recently diagnosed and were fast progressors.

There was a much higher viral load at diagnosis in patients who turned out to have the CRF19_cpx subtype; in the 6 patients with this subtype whose viral load was measured at diagnosis, their viral load averaged over 1 million copies/mL.

Testing 3 Years After Infection

The 73 patients who were recently diagnosed were then evaluated prospectively, meaning that their viral load, CD4 count, and CD4 percentage were measured again at the 3-year mark. The chronically infected patients were retrospectively evaluated to find their viral load and CD4 count and percentage at the test nearest to their reaching an AIDS-defining criterion (i.e., around the year 2008).

At this point in time there was no difference in the viral load between the fast progressors and the chronically infected patients, who had viral loads averaging 50,000 copies/mL; the viral load in patients with CRF19-cpx was not significantly higher either. The slow progressors had a viral load 10 times lower -- 5000 copies/mL -- but no longer undetectable, and this was not statistically different from other patients. The average CD4 count in the fast progressors at this time was 189 cells/mm3, in the chronically infected patients 212 cells/mm3, and in the slow progressors 501 cells/mm3.

The researchers measured a number of different cytokines, or soluble immune-modulating molecules indicative of inflammation. They found very low levels of most of them in the slow progressor group, and high levels in both groups with an AIDS diagnosis. The only significant difference between the fast progressors and the chronically infected group was that the former had higher levels of the cytokine called RANTES. This is particularly interesting as RANTES is a natural blocker of HIV infection, upon which CCR5 inhibitors such as maraviroc (Selzentry) were based.

The researchers found that oral candidiasis was the only one of a range of sexually (or sometimes sexually) transmitted infections associated with being a fast progressor. While 23% of this group had oral candidiasis, none of the chronically infected patients with AIDS and less than 5% of the slow progressors had it.

As noted above, HIV viral subtype varied by group. Slow progressors either had subtype B (42%), one of several different B/G recombinant viruses (37%), or a recombinant called CRF18_cpx (11%); in the remainder, subtype could not be determined. Among the chronically infected patients with AIDS, the proportions were roughly similar except for a few subtype C cases too. Among the fast progressors, 17% of them were, as already noted, the only people with the CRF19_cpx virus. In addition, far fewer had subtype B -- again, only 17%. About one-third of them had the B/G recombinant virus and 10% had subtype C. So while this hardly indicates that the CRF19_cpx virus was the cause of all fast progression, it might indicate that its recent expansion into the population could be linked to an apparent increase in fast progression.

The researchers also mentioned the tropism of the virus. Different varieties of HIV prefer to infect different immune system cells, and tropism measures whether HIV prefers to link to a cell coreceptor called CCR5, a different one called CXCR4, or is able to use either one. Tropism is complex -- see this article for a good explanation. CCR5-tropic viruses are easily transmitted and proliferate well, but cause relatively limited immune system damage and slow progression, whereas CXCR4-tropic viruses (and mixed-tropism ones) are rarely transmitted and replicate less well, but cause devastating immune damage. Part of the causation of AIDS is that HIV tends to mutate into versions that can use the CXCR4 receptor within the body, and once a certain proportion of virus becomes CXCR4-tropic, very fast immune decline results.

In this study, none of the slow progressors had virus that exclusively used CXCR4; this compared with 16% of fast progressors and 19% of chronically infected patients with AIDS. Conversely, 71% of slow progressors had virus that exclusively used CCR5, compared with 50% of the chronically infected patients and 46% of fast progressors.

However, these tropism differences were not statistically significant, and in fact the researchers found that the part of the CRF19_cpx virus genome responsible for binding to cells was from HIV subtype A, which is not associated with rapid switching to CXCR4 tropism.

While viral load at the 3-year sampling mark was not significantly different between the groups, if the production of just one component of HIV -- its protease protein -- was measured, the researchers found that patients with CRF19_cpx had protease levels nearly 100 times higher than others. The gene for protease in the recombinant virus comes from the more virulent subtype D. Protease is responsible for part of the process of manufacturing new viral particles, so this could explain CRF19_cpx’s association with higher viral load.

Possible Conclusions

The researchers' hypothesis is this: CRF19_cpx initially replicates at very high levels -- as that 6 million-copy viral load shows. At this time it will be CCR5-tropic and infecting cells with that receptor. But the body will manufacture huge amounts of the protein RANTES as a defense against cellular invasion -- as shown by its higher levels in people with CRF19_cpx.

This protein blocks CRF19_cpx from infecting any more cells via this route. So the virus is forced early on to change to a CXCR4-tropic strain. This type then reproduces more slowly than before -- hence the lack of difference in viral load at the 3-year mark -- but is much more pathogenic. That is the hypothesis, anyway. But as the researchers point out, they did not measure the entire gene sequence of CRF19_cpx, so it could have other characteristics they missed.

Nonetheless -- even though CRF19_cpx is still very much a minority variant in a minority of people -- this is the first time a variety of HIV has been found to be exclusively associated with people who progress rapidly to AIDS. However, there is no evidence that CRF19_cpx is more susceptible to becoming drug resistant, and ART should work against it just as well. But this is a reminder of HIV’s genetic adaptability, and that the timespan between HIV infection and symptomatic AIDS may be shorter than we think in some people.

2/20/15

Reference

V Kouri, R Khouri, Y Alemán, et al. CRF19_cpx is an evolutionary fit HIV-1 variant strongly associated with rapid progression to AIDS in Cuba.EBioMedicine. January 28, 2015 (in press).

Other Source

Catholic University of Leuven in Belgium (KU Leuven). An Aggressive Form of HIV Uncovered in Cuba. Press release. February 12, 2015.